Arginine repressor deficient strain of coryneform bacterium and method for producing L-arginine

ABSTRACT

L-Arginine is produced by culturing a coryneform bacterium in which an arginine repressor involved in L-arginine biosynthesis is deleted by disrupting a gene coding for the repressor, and which has L-arginine producing ability in a medium to produce and accumulate L-arginine in the medium, and collecting the L-arginine from the medium.

FIELD OF THE INVENTION

The present invention relates to a coryneform bacterium having anability to produce L-arginine and a method for producing L-arginineusing the bacterium. L-arginine is an industrially useful amino acid asan ingredient of liver function promoting agents, amino acid infusions,comprehensive amino acid pharmaceuticals and so forth.

DESCRIPTION OF THE RELATED ART

Conventional L-arginine production by fermentation has been performed byutilizing wild-type strains of coryneform bacteria; coryneform bacteriaresistant to certain agents including sulfa drugs, 2-thiazolealanine,â-amino-â-hydroxyvaleric acid and the like; coryneform bacteriaexhibiting auxotrophy for L-histidine, L-proline, L-threonine,L-isoleucine, L-methionine, or L-tryptophan in addition to theresistance to 2-thiazolealanine (Japanese Patent Laid-open No.54-44096); coryneform bacteria resistant to ketomalonic acid,fluoromalonic acid, or monofluoroacetic acid (Japanese Patent Laid-openNo. 57-18989); coryneform bacteria resistant to argininol (JapanesePatent Laid-open No. 62-24075); coryneform bacteria resistant toX-guanidine (X represents a derivative of fatty acid or aliphatic chain,Japanese Patent Laid-open No. 2-186995) or the like.

On the other hand, there have also been disclosed methods for producin gL-arginine utilizing recombinant DNA techniques. That is, there has beendisclosed a method for producing L-arginine by utilizing a microorganismbelonging to the genus Corynebacterium or Brevibacterium which is madeto harbor a recombinant DNA comprising a vector DNA and a DNA fragmentcontaining genes for acetylornithine deacetylase, N-acetylglutamicacid-â-semialdehyde dehydrogenase, N-acetyl glutamokinase, andargininosuccinase derived from a microorganism belonging to the genusEscherichia (Japanese Patent Publication No. 5-23750).

Further, as for coryneform bacteria, it has been elucidated thatsynthesis of some enzymes of the L-arginine biosynthetic system isrepressed by L-arginine. Furthermore, it was reported that, while someof enzymes of L-arginine biosynthetic system were repressed byL-arginine, the repression of these enzymes by L-arginine was canceledin mutant strains of coryneform bacteria showing improved L-arginineaccumulation amounts (Agric. Biol. Chem., 43(1), 105, 1979).

Meanwhile, as for Escherichia coli, a repressor of L-argininebiosynthetic system and a gene coding for the repressor were identified(Proc. Natl. Acad. Sci. U.S.A. (1987), 84(19), 6697-701), and bindinginteractions of the repressor protein and various genes of L-argininebiosynthetic system were also investigated (Proc. Natl. Acad. Sci.U.S.A. (1987), 84(19), 6697-701, J. Mol. Biol. (1992), 226, 367-386).

However, any repressor proteins of the L-arginine biosynthetic systemhave not been identified in coryneform bacteria. While a nucleotidesequence of the repressor protein gene (argR) and an amino acid sequenceassumed to be encoded thereby are registered in a gene database, GenBank(AF049897), the gene is considered to be designated argR because of thehomology between the aforementioned amino acid sequence and knownarginine repressors.

SUMMARY OF THE INVENTION

As described above, although a repressor protein of the L-argininebiosynthetic system of coryneform bacteria and a gene thereof areexpected, the repressor protein itself has not been identified and itsfunctions and so forth are not elucidated at all. Therefore, an objectof the present invention is to identify the repressor of the L-argininebiosynthesis in coryneform bacteria, and improve L-arginine productivityof coryneform bacteria.

The inventors of the present invention isolated a homologue of the generegistered as argR in the gene database (GenBank accession AF049897)from a coryneform bacterium, and found that, if this gene was amplifiedin coryneform bacteria, L-arginine producing ability was decreased, andon the other hand, if the gene was disrupted, the L-arginine producingability was improved, to confirm that the L-arginine biosynthesis isrepressed by a repressor in coryneform bacteria like Escherichia coliand the aforementioned gene registered as argR codes for the repressor.Thus, the present invention was accomplished.

That is, the present invention provides the followings.

(1) A coryneform bacterium in which an arginine repressor does notfunction in a normal manner, and which has L-arginine producing ability.

(2) The coryneform bacterium according to (1), wherein the argininerepressor does not function in a normal manner due to disruption of agene coding for the arginine repressor on a chromosome of the bacterium.

(3) The coryneform bacterium according to (2), wherein the argininerepressor has the amino acid sequence shown in SEQ ID NO: 18 or an aminoacid sequence showing homology to the amino acid sequence.

(4) A method for producing L-arginine, which comprises culturing acoryneform bacterium according to any one of (1) to (3) in a medium toproduce and accumulate L-arginine in the medium, and collecting theL-arginine from the medium.

In the present invention, the “arginine repressor” refers to a proteinthat has an effect of repressing the L-arginine biosynthesis, and ifexpression amount of the gene that codes for the protein increases incoryneform bacteria, L-arginine producing ability will be reduced, andif the expression amount decreases or the protein disappears, theL-arginine producing ability will be improved. Hereafter, the genecoding for the arginine repressor is also called argR gene. Further, the“L-arginine producing ability” used in the present invention refers toan ability of the microorganism of the present invention to accumulateL-arginine in a medium, when it is cultured in the medium.

According to the present invention, L-arginine producing ability ofcoryneform bacteria having the L-arginine producing ability can beimproved.

BRIEF EXPLANATION OF THE DRAWINGS

FIG. 1 shows the construction process of plasmid pK1.

FIG. 2 shows the construction process of plasmid pSFK6.

FIG. 3 shows the construction process of plasmid pSFKT2.

Hereafter, the present invention will be explained in detail.

The microorganism of the present invention is a coryneform bacteriumhaving L-arginine producing ability, in which arginine repressor doesnot function in a normal manner. The coryneform bacterium of the presentinvention may be a microorganism having the L-arginine producing abilitybecause an arginine repressor does not function in a normal manner init, or a microorganism bred so that the arginine repressor should notfunction in a normal manner in it. Alternatively, it may be amicroorganism that is bred so that the arginine repressor should notfunction in a normal manner in it and then imparted with the L-arginineproducing ability.

The coryneform bacteria include bacteria having been hitherto classifiedinto the genus Brevibacterium but united into the genus Corynebacteriumat present (Int. J. Syst. Bacteriol., 41, 255 (1981)), and includebacteria belonging to the genus Brevibacterium closely relative to thegenus Corynebacterium. Examples of such coryneform bacteria include thefollowings.

Corynebacterium acetoacidophilum

Corynebacterium acetoglutamicum

Corynebacterium alkanolyticum

Corynebacterium callunae

Corynebacterium glutamicum

Corynebacterium lilium (Corynebacterium glutamicum)

Corynebacterium melassecola

Corynebacterium thermoaminogenes

Corynebacterium herculis

Brevibacterium divaricatum

(Corynebacterium glutamicum)

Brevibacterium flavum (Corynebacterium glutamicum)

Brevibacterium immariophilum

Brevibacterium lactofermentum

(Corynebacterium glutamicum)

Brevibacterium roseum

Brevibacterium saccharolyticum

Brevibacterium thiogenitalis

Brevibacterium album

Brevibacterium cerinum

Microbacterium ammoniaphilum

While the coryneform bacteria that have the L-arginine-producing abilityare not particularly limited so long as they have theL-arginine-producing ability, they include, for example, wild-typestrains of coryneform bacteria; coryneform bacteria resistant to certainagents including sulfa drugs, 2-thiazolealanine,α-amino-β-hydroxyvaleric acid and the like; coryneform bacteriaexhibiting auxotrophy for L-histidine, L-proline, L-threonine,L-isoleucine, L-methionine, or L-tryptophan in addition to theresistance to 2-thiazolealanine (Japanese Patent Laid-open No.54-44096); coryneform bacteria resistant to ketomalonic. acid,fluoromalonic acid, or monofluoroacetic acid (Japanese Patent Laid-openNo. 57-18989); coryneform bacteria resistant to argininol (JapanesePatent Laid-open No. 62-24075); coryneform bacteria resistant toX-guanidine (X represents a derivative of fatty acid or aliphatic chain,Japanese Patent Laid-open No. 2-186995) and the like.

Specifically, the following strains can be exemplified.

Brevibacterium flavum AJ11169 (BP-6892)

Corynebacterium glutamicum AJ12092 (FERM BP-6906)

Brevibacterium flavum AJ11336 (FERM BP-6893)

Brevibacterium flavum AJ11345 (FERM BP-6894)

Corynebacterium glutamicum AJ12430 (FERM BP-2228)

The AJ11169 strain and the AJ12092 strain are the 2-thiazolealanineresistant strains mentioned in Japanese Patent Laid-open No. 54-44096,the AJ11336 strain is the strain having argininol resistance andsulfadiazine resistance mentioned in Japanese Patent Publication No.62-24075, the AJ11345 strain is the strain having argininol resistance,2-thiazolealanine resistance, sulfaguanidine resistance, and exhibitinghistidine auxotrophy mentioned in Japanese Patent Publication No.62-24075, and the AJ12430 strain is the strain having octylguanidineresistance and 2-thiazolealanine resistance mentioned in Japanese PatentLaid-open No. 2-186995.

AJ11169 was deposited on Aug. 3, 1977 in National Institute ofBioscience and Human Technology, Agency of Industrial Science andTechnology, Ministry of International Trade and Industry (currentlyNational Institute of Bioscience and Human Technology, NationalInstitute of Advanced Industrial Science and Technology, Ministry ofEconomy, Trade and Industry)(zip code: 305-8566, 1-3 Higashi 1-Chome,Tsukuba-shi, Ibaraki-ken, Japan), as deposition number of FERM P-4161,and transferred from the original deposit to international deposit basedon Budapest Treaty on Sep. 27, 1999, and has been deposited asdeposition number of FERM BP-6892.

AJ12092 was deposited on September 29, 1983 in National Institute ofBioscience and Human Technology, Agency of Industrial Science andTechnology, Ministry of International Trade and Industry, as depositionnumber of FERM P-7273, and transferred from the original deposit tointernational deposit based on Budapest Treaty on Oct. 1, 1999, and hasbeen deposited as deposition number of FERM BP-6906.

AJ11336 was deposited on Apr. 25, 1979 in National Institute ofBioscience and Human Technology, Agency of Industrial Science andTechnology, Ministry of International Trade and Industry, as depositionnumber of FERM P-4939, and transferred from the original deposit tointernational deposit based on Budapest Treaty on Sep. 27, 1999, and hasbeen deposited as deposition number of FERM BP-6893.

AJ11345 was deposited on Apr. 25, 1979 in National Institute ofBioscience and Human Technology, Agency of Industrial Science andTechnology, Ministry of International Trade and Industry, as depositionnumber of FERM P-4948, and transferred from the original deposit tointernational deposit based on Budapest Treaty on Sep. 27, 1999, and hasbeen deposited as deposition number of FERM BP-6894.

AJ12430 was deposited on Dec. 26, 1988 in National Institute ofBioscience and Human Technology, Agency of Industrial Science andTechnology of Ministry, International Trade and Industry based onBudapest Treaty, as deposition number of FERM BP-2228.

The coryneform bacterium whose arginine repressor does not function in anormal manner can be obtained by modifying its argR gene so that theactivity of the arginine repressor should be reduced or eliminated, orthe transcription of the argR gene should be reduced or eliminated. Sucha coryneform bacterium can be obtained by, for example, replacing thechromosomal argR gene with an argR gene that does not function in anormal manner (occasionally referred to as “disrupted argR gene”hereinafter) through, for example, homologous recombination based ongenetic recombination techniques (Experiments in Molecular Genetics,Cold Spring Harbor Laboratory Press (1972); Matsuyama, S. and Mizushima,S., J. Bacteriol., 162, 1196 (1985)).

In the homologous recombination, when a plasmid carrying a sequenceexhibiting homology with a chromosomal sequence or the like isintroduced into a corresponding bacterial cell, recombination occurs ata site of the homologous sequence at a certain frequency, and thus theintroduced plasmid as a whole is integrated into the chromosome. Then,by causing recombination again at the site of the homologous sequence inthe chromosome, the plasmid may be removed from the chromosome. However,depending on the position at which the recombination is caused, thedisrupted gene may remain on the chromosome, while the original normalgene may be removed from the chromosome together with the plasmid. Byselecting such a bacterial strain, a bacterial strain in which thenormal argR gene is replaced with a disrupted argR gene can be obtained.

Such a gene disruption technique based on the homologous recombinationhas already been established, and a method utilizing a linear DNA,method utilizing temperature sensitive plasmid or the like can be usedtherefor. The argR gene can also be disrupted by using a plasmid thatcontains the argR gene inserted with a marker gene such as drugresistance gene, and cannot replicate in a target cell of the coryneformbacterium. That is, in a transformant that has been transformed withsuch a plasmid and hence acquired drug resistance, the marker gene isintegrated in a chromosome DNA. It is likely that this marker gene hasbeen integrated by homologous recombination of the argR gene present atthe both sides of the marker with the argR on the chromosome, andtherefore a gene-disrupted strain can efficiently be selected.

Specifically, a disrupted argR gene used for the gene disruption can beobtained by deletion of a certain region of argR gene by means ofdigestion with restriction exzyme(s) and religation; by insertion ofanother DNA fragment (marker gene etc.) into the argR gene, byintroducing substitution, deletion, insertion, addition or inversion ofone or more nucleotides in a nucleotide sequence of coding region ofargR gene, its promoter region or the like by means of site-specificmutagenesis (Kramer, W. and Frits, H. J., Methods in Enzymology, 154,350 (1987)) or treatment with a chemical reagent such as sodiumhyposulfite and hydroxylamine (Shortle, D. and Nathans, D., Proc. Natl.Acad. Sci. U.S.A., 75, 270(1978)) or the like, so that the activity ofthe encoded repressor should be reduced or eliminated, or transcriptionof the argR gene should be reduced or eliminated. Among theseembodiments, a method utilizing deletion of a certain region of the argRgene by digestion with a restriction enzyme and religation, or insertionof another DNA fragment into the argR gene is preferred in view ofreliability and stability.

A plasmid for the argR gene disruption can be produced by performing PCR(polymerase chain reaction) using a plasmid containing the argR gene andits flanking regions as a template and primers corresponding theterminal portions or franking regions of the argR gene to amplify aportion except for an internal portion or the whole portion of the argRgene, and cyclizing the obtained amplified product. In the examplesmentioned hereinafter, the argR gene was disrupted by this method.

The argR gene can be obtained from a chromosomal DNA of a coryneformbacterium by PCR using oligonucleotides prepared based on knownnucleotide sequences of the argR gene as primers. The argR gene can alsobe obtained from a chromosome DNA library of a microorganism which has apurine operon by a hybridization technique using an oligonucleotideprepared based on a known nucleotide sequence of the argR gene as aprobe. For the purpose of the present invention, because the argR geneis used for preparing a disrupted argR gene, it is not necessarilyrequired to contain the full length, and it may contain a lengthrequired to cause gene disruption.

The origin of the argR gene is not particularly limited, so long as ithas such a degree of homology that it should cause homologousrecombination with the argR gene of coryneform bacteria. Specifically,the argR gene of the Brevibacterium flavum, which has the nucleotidesequence shown in SEQ ID NO: 17, and the argR gene of Corynebacteriumglutamicum (GenBank accession AF049897) can be mentioned as the argRgenes of coryneform bacteria. These argR genes are highly homologous,and it is considered that even an argR gene of coryneform bacterium of agenus or species different from that of a coryneform bacterium of whichargR gene is to be disrupted may also be used for the gene disruption.

In the present invention, the amino acid sequence shown in SEQ ID NO: 18or an amino acid sequence exhibiting homology to the amino acid sequencemeans an amino acid sequence that is encoded by an argR gene having sucha degree of homology that it should cause homologous recombination withthe argR gene coding to the amino acid sequence shown in SEQ ID NO: 18(for example, an argR gene having the nucleotide sequence shown in SEQID NO: 17).

As the primers used for PCR, any primers that allow amplification of theargR gene can be used. Specific examples thereof include theoligonucleotides having the nucleotide sequences shown in SEQ ID NOS: 19and 20.

Further, examples of marker gene include drug resistance genes such as akanamycin resistance gene. A kanamycin resistance gene can be obtainedby PCR amplification from a known plasmid containing a kanamycinresistance gene of Streptococcus faecalis, for example, pDG783(Anne-Marie Guerout-Fleury et al., Gene, 167, 335-337 (1995)).

When a drug resistance gene is used as the marker gene, an argRgene-disrupted strain can be obtained by inserting the drug resistancegene into a suitable site of the argR gene carried by a plasmid,transforming a microorganism with the plasmid, and selecting a drugresistant transformant. Disruption of argR gene on a chromosome can beconfirmed by analyzing the argR gene or the marker gene on thechromosome by Southern blotting, PCR, or the like. Integration of thekanamycin resistance gene into a chromosomal DNA can be confirmed by PCRusing primers that allow amplification of the kanamycin resistance gene(e.g., oligonucleotides having nucleotide sequences shown in SEQ ID NOS:1 and 2).

L-arginine can be efficiently produced by culturing a coryneformbacterium having L-arginine producing ability obtained as describedabove, in which an arginine repressor does not function in a normalmanner, in a medium to produce and accumulate L-arginine in the medium,and collecting the L-arginine from the medium.

The medium to be used may be selected from well-known mediaconventionally used for fermentative production of amino acids utilizingmicroorganisms. That is, it may be a usual medium that contains a carbonsource, nitrogen source, inorganic ions, and other organic ingredientsas required.

As the carbon source, there can be used saccharides such as glucose,sucrose, lactose, galactose, fructose or starch hydrolysate, alcoholssuch as glycerol or sorbitol, or organic acids such as fumaric acid,citric acid or succinic acid.

As the nitrogen source, there can be used inorganic ammonium salts suchas ammonium sulfate, ammonium chloride or ammonium phosphate, organicnitrogen such as soybean protein hydrolysate, ammonia gas, aqueousammonia and so forth.

It is desirable to add required substances such as vitamin B₁ andL-homoserine, yeast extract and so forth to the medium in appropriateamounts as organic trace nutrients. Other than the above, potassiumphosphate, magnesium sulfate, iron ion, manganese ion and so forth areadded in small amounts as required.

The culture is preferably carried out under an aerobic condition for 1-7days. The culture temperature is preferably controlled to be 24° C. to37° C., and pH is preferably controlled to be 5-9 during the culture.Inorganic or organic, acidic, alkaline substances, or ammonia gas and soforth can be used for pH adjustment. L-arginine can be collected fromthe fermentation broth usually by a combination of well-known techniquessuch as ion exchange resin techniques and other techniques.

BEST MODE FOR CARRYING OUT THE INVENTION

Hereafter, the present invention will be explained more specificallywith reference to the following examples.

EXAMPLE 1 Constructions of Shuttle Vector for Escherichia coli andcoryneform Bacteria and Temperature Sensitive Vector

First, a vector for introducing an argR gene into coryneform bacteriaand a temperature sensitive vector for producing an argR deficientstrain of coryneform bacterium were constructed.

<1> Construction of Vector Having Drug Resistance Gene of Streptococcusfaecalis

The kanamycin resistance gene of Streptococcus faecalis was amplified byPCR from a known plasmid containing the gene. The nucleotide sequence ofthe kanamycin resistance gene of Streptococcus faecalis has already beenelucidated (Trieu-Cuot, P. and Courvalin, P.: Gene, 23 (3), 331-341(1983)). Based on this sequence, the primers shown in SEQ ID NOS: 1 and2 were synthesized, and PCR was performed by using pDG783 (Anne-MarieGuerout-Fleury, et al., Gene, 167, 335-337 (1995)) as a template toamplify a DNA fragment containing the kanamycin resistance gene and itspromoter.

The aforementioned DNA fragment was purified by using SUPREC02 producedby Takara Shuzo Co., Ltd., and then completely digested with restrictionenzymes HindIII and HincII to be blunt-ended. The blunt-ending wasperformed by using Blunting Kit produced by Takara Shuzo Co., Ltd. ThisDNA fragment was mixed with a DNA fragment obtained by purification andblunt-ending of an amplification product of PCR performed by using theprimers shown in SEQ ID NOS: 3 and 4 and pHSG399 (see S. Takeshita, etal.: Gene, 61, 63-74 (1987)) as a template, and ligated both fragments.The ligation was performed by using DNA Ligation Kit Ver. 2 produced byTakara Shuzo Co., Ltd. Competent cells of Escherichia coli JM109(produced by Takara Shuzo Co., Ltd.) were transformed with the ligatedDNA, plated on L medium (10 g/L of Bacto trypton, 5 g/L of Bacto yeastextract, 5 g/L of NaCl, 15 g/L of agar, pH 7.2) containing 10 μg/ml ofIPTG (isopropyl-â-D-thiogalactopyranoside), 40 μg/ml of X-Gal(5-bromo-4-chloro-3-indolyl-â-D-galactoside) and 25 μg/ml of kanamycin,and cultured overnight. The emerged blue colonies were picked up, andseparated into single colonies to obtain transformant strains.

Plasmids were prepared from the transformant strains by the alkalimethod (Seibutsu Kogaku Jikkensyo (Text for Bioengineering Experiments),Edited by the Society for Bioscience and Bioengineering, Japan, p.105,Baifukan, 1992), and restriction maps were prepared. One having arestriction map equivalent to that of FIG. 1 was designated as pKl. Thisplasmid is stably harbored in Escherichia coli, and imparts kanamycinresistance to a host. Moreover, since it contains the lacZ′ gene, it issuitably used as a cloning vector.

<2> Construction of Shuttle Vector pSFK6

As a material for obtaining a temperature sensitive replication controlregion, a plasmid vector autonomously replicable in both of Escherichiacoli cells and coryneform bacteria cells was prepared. The plasmidpAM330 extracted from Brevibacterium lactofermentum ATCC13869 [seeJapanese Patent Publication Laid-open (Kokai) No. 58-67699] wascompletely digested with a restriction enzyme HindIII, and blunt-ended.This fragment was ligated to a fragment obtained by completely digestingthe aforementioned pK1 with a restriction enzyme BsaAI. Brevibacteriumlactofermentum ATCC13869 was transformed with the ligated DNA. Thetransformation was performed by the electric pulse method [see JapanesePatent Publication Laid-open (Kokai) No. 2-207791]. Transformants wereselected on an M-CM2B plate (10 g/L of polypeptone, 10 g/L of yeastextract, 5 g/L of NaCl, 10 μg/L of biotin, 15 g/L of agar, pH 7.2)containing 25 μg/ml of kanamycin. After cultivation for 2 days, colonieswere picked up, and separated into single colonies to obtain thetransformants. Plasmid DNAs were prepared from the transformants, andrestriction maps were prepared. One having the same restriction map asthat of FIG. 2 was designated as pSFK6. This plasmid is autonomouslyreplicable in both of Escherichia coli and coryneform bacteria, andimparts kanamycin resistance to a host.

<3> Construction of a Plasmid Having Temperature Sensitive ReplicationControl Region

pSFK6 was treated with hydroxylamine in vitro. The hydroxylaminetreatment was performed according to a known method [see, for example,G. O. Humpherys, et al., Molec. Gen. Genet., 145, 101-108 (1976)]. DNAundergone the treatment was collected and used for transformation ofBrevibacterium lactofermentum ATCC13869 strain. The transformants wereselected at a low temperature (25° C.) on a CM2B plate containing 25μg/ml of kanamycin. The appeared transformants were replicated to asimilar selection plate, and cultured at an elevated temperature (34°C.). One strain that could not grow on the selection plate containingkanamycin at the elevated temperature was obtained. From this strain, aplasmid was recovered and designated as p48K.

<4> Determination of Nucleotide Sequence of Temperature SensitiveReplication Control Region

Nucleotide sequences of replication control region segments in theplasmid pSFK6 having a wild-type replication control region and theplasmid p48K having a temperature sensitive replication control regionwere determined. The nucleotide sequences were determined on a fullyautomatic sequencer, ABI310 (ABI), by using DNA Sequencing Kit from ABI.As a result, it was found that there were 6 nucleotide substitutionsbetween the wild-type replication control region and the temperaturesensitive replication control region. The nucleotide sequence of thetemperature sensitive replication control region segment contained inpSFK6 (derived from full sequence of pAM330), which functions incoryneform bacteria, is shown in SEQ ID NO: 5, and the nucleotidesequence of the temperature sensitive replication control region segmentcontained in p48K, which functions in coryneform bacteria, is shown inSEQ ID NO: 7. Further, the amino acid sequences encoded by ORFscontained in these nucleotide sequences are shown in SEQ ID NOS: 6 and8. In the temperature sensitive replication control region, the 1255th Cis mutated to T, the 1534th C to T, the 1866th G to A, the 2058th G toA, the 2187th C to T and 3193rd G to A. Among these, only the mutationat 1534th position is accompanied by an amino acid mutation, and causessubstitution of serine for proline.

<5> Construction of Shuttle Vectors Having Temperature SensitiveMutation

Each one of the six mutations of p48K was introduced into a shuttlevector pSFK6 (see FIG. 3). The introduction of the mutations wasperformed by a known method [Mikaelian,. I., Sergeant, A., Nucleic AcidsRes., 20, 376 (1992)]. Specific procedure will be mentioned below. Inorder to introduce the mutation of 1534th C to T, PCR was performed byusing a combination of the primers shown in SEQ ID NOS: 9 and 10(primers 9 and 10), and a combination of the primers shown in SEQ IDNOS: 11 and 12 (primers 11 and 12), and pAM330 as a template. Each ofthe obtained amplification products was purified by subjecting them toagarose gel electrophoresis, and collecting them from the gel. Thecollection of the DNA fragments from the gel was performed by usingEASYTRAP Ver.2 (Takara Shuzo Co., Ltd.). The purified DNAs were mixed ina molar ratio of 1:1, and used as a template for PCR performed by usingthe primers shown SEQ ID NOS: 13 and 14 (primers 13 and 14). Theamplification product was fully digested with a restriction enzyme MluI,and subjected to agarose gel electrophoresis to recover a DNA fragmentof about 3.2 kb. Similarly, pSFK6 was also completely digested with arestriction enzyme MluI, and subjected to agarose gel electrophoresis torecover a DNA fragment of about 3.8 kb. The obtained DNA fragments weremixed and ligated, and used to transform competent cells of Escherichiacoli JM109 (Takara Shuzo Co., Ltd.). The cells were applied on L mediumcontaining 25 μg/ml of kanamycin, and cultured overnight. The appearedcolonies were picked up, and isolated single colonies to obtaintransformant strains. A plasmid was prepared from the transformantstrains by the alkaline method, and the nucleotide sequence of theplasmid was determined to confirm that 1534th C in the sequence shown inSEQ ID NO: 5 was mutated to T. This plasmid was designated as pSFKT2(FIG. 3).

EXAMPLE 2 Cloning of argR Gene and Amplification Effect Thereof incoryneform Bacteria

PCR was performed by using chromosome DNA of the Brevibacterium flavumwild strain 2247 (AJ14067) as a template and the oligonucleotides havingthe nucleotide sequences shown in SEQ ID NO: 15 (sequence of thenucleotide numbers 1717-1741 in SEQ ID NO: 17) and SEQ ID NO: 16(sequence complementary to the sequence of the nucleotide numbers2386-2362 in SEQ ID NO: 17) as primers (Primers 15 and 16). PCR wasperformed for 30 cycles with each cycle consisting of reactions at 98° Cfor 10 seconds, 58° C. for 1 minute and 72° C. for 3 minutes by usingPyrobest DNA polymerase (Takara Shuzo Co., Ltd.). The obtained amplifiedfragment was inserted into the SmaI site of the shuttle vector pSFK6obtained in Example 1 to obtain plasmid pWR autonomously replicable incoryneform bacteria.

In order to investigate the amplification effect of argR gene inL-arginine producing coryneform bacteria, pWR was introduced into theAJ113455 strain (FERM BP-6894), which is an L-arginine producer ofBrevibacterium flavum. The plasmid was introduced by the electric pulsemethod (Japanese Patent Laid-open No. 2-207791). A.transformant wasselected as a kanamycin resistant strain on CM2G agar medium (containing5 g of glucose, 5 g of NaCl and 15 g of agar in 1 L of pure water, pH7.2) containing 25 μg/ml of kanamycin to obtain AJ11345/pWR. As acontrol, pSFK6 was similarly introduced into the AJ113455 strain toobtain a transformant AJ11345/pSFK6.

Each of the aforementioned strains was plated on an agar mediumcontaining 0.5 g/dl of glucose, 1 g/dl of polypeptone, 1 g/dl of yeastextract and 0.5 g/dl of NaCl, and cultured at 31.5° C. for 20 hours. Oneplatunum loop of the obtained cells were inoculated into a mediumcontaining 4 g/dl of glucose, 6.5 g/dl of ammonium sulfate, 0.1 g/dl ofKH₂PO₄, 0.04 g/dl of MgSO₄, 0.001 g/dl of FeSO₄, 0.001 g/dl of MnSO₄, 5pg/dl of vitamin B₁, 5 μg/dl of biotin and soybean protein hydrolysate(45 mg/dl as N amount), and cultured in a flask at 31.5° C. for 50 hourswith shaking. Accumulation amount of L-arginine (concentration, g/dl) ineach culture broth was measured. The results are shown in Table 1. As aresult, the argR-amplified strain hardly accumulated L-arginine. Thisdemonstrated that the argR gene product functioned as an argininerepressor. TABLE 1 Strain L-Arginine accumulation amount (g/dl)AJ11345/pSFK6 1.3 AJ11345/pWR 0.2

The result of nucleotide sequencing for the inserted fragment cloned inpWR is shown in SEQ ID NO: 17. An amino acid sequence that may beencoded by that nucleotide sequence is shown in SEQ ID NO: 18.

EXAMPLE 3 Construction of argR-Disrupted Strain of coryneform Bacteriumand Effect of Deletion of arginine Repressor

<1>Construction of plasmid for argR Disruption

PCR was performed by using chromosome DNA of a wild strain ofBrevibacterium flavum, 2247 strain (AJ14067), as a template and theoligonucleotides having the nucleotide sequences shown in SEQ ID NO: 19(sequence of the nucleotide numbers 4-28 in SEQ ID NO: 17) and SEQ IDNO: 20 (sequence complementary to the sequence of the nucleotide numbers4230-4211 in SEQ ID NO: 17) as primers (Primers 19 and 20). PCR wasperformed for 30 cycles with each cycle consisting of reactions at 98°C. for 10 seconds, 58° C. for 1 minute and 72° C. for 3 minutes by usingPyrobest DNA polymerase (Takara Shuzo Co., Ltd.). The obtained amplifiedfragment was inserted into the SmaI site in a multicloning site ofcloning vector pHSG399.

In order to delete the whole ORF considered to encode the argininerepressor from the inserted DNA fragment, PCR was performed by using theoligonucleotides having the nucleotide sequences shown in SEQ ID NO: 21(sequence of the nucleotide numbers 2372-2395 in SEQ ID NO: 17) and SEQID NO: 22 (sequence complementary to the sequence of the nucleotidenumbers 1851-1827 in SEQ ID NO: 17) as primers (Primers 21 and 22) andpHSG399 inserted with the amplified fragment as a template. pssER wasconstructed by self-ligation of the PCR product.

Then, a fragment obtained by digesting pssER with restriction enzymesSmaI and SalI and the temperature sensitive plasmid pSFKT2 obtained inExample 1 and digested with SmaI and SalI were ligated to obtain plasmidpssERT for argR disruption whose autonomous replication ability incoryneform bacteria became temperature sensitive.

<2> Construction of arginine Repressor Deficient Strain of coryneformbacterium by Homologous Recombination

The plasmid pssERT obtained as described above was introduced into theBrevibacterium lactofermentum AJ13029 strain (FERM BP-5189). The plasmidwas introduced by the electric pulse method (Japanese Patent Laid-openNo. 2-207791). Because autonomous replication ability of this plasmid istemperature sensitive in Brevibacterium lactofermentum, only strains inwhich this plasmid was incorporated into the chromosome by homologousrecombination could be selected as kanamycin resistant strains at 34°C., which was a temperature that did not allow replication of theplasmid. Strains in which the plasmid for argR disruption wasincorporated into a chromosome were selected as kanamycin resistantstrains on a CM2G plate (containing 10 g/L of polypeptone, 10 g/L ofyeast extract, 5 g/L of glucose, 5 g/L of NaCl and 15 g/L of agar in 1 Lof water, pH 7.2) containing 25 μg/ml of kanamycin. At this stage, thenormal argR gene derived from the chromosome and the argR gene derivedfrom the plasmid in which OFR was deleted were present in tandem at theboth sides of the plasmid portion on the chromosome.

Then, the recombinant strains were allowed to cause homologousrecombination again, and strains that became kanamycin sensitive at 34°C., which was a temperature that did not allow the plasmid replication,were selected as strains in which one of the argR genes was deleted.These strains include strains in which the normal argR gene remained onthe chromosome and strains in which the disrupted argR gene remained onthe chromosome. From these strains, a strain having only the disruptedargR gene was selected. An argR gene on the chromosome is determined tobe the disrupted type by preparing chromosome of a strain that becamekanamycin sensitive at 34° C., performing PCR utilizing the chromosomeas a template and the oligonucleotides having the nucleotide sequencesshown in SEQ ID NOS: 19 and 20 as primers (Primers 19 and 20), andconfirming that the PCR product was shorter by about 600 bp than thatobtained by similarly performing PCR utilizing chromosome derived fromthe parent strain as a template.

Direct sequencing of the PCR product of the argR-disrupted strainselected as described above was performed to confirm that the argR genewas disrupted as desired, and thus AJ13029AR stain was obtained.<3>Production of L-arginine with argR-disrupted strain The AJ13029ARstrain was plated on an agar medium containing 0.5 g/dl of glucose, 1g/dl of polypeptone, 1 g/dl of yeast extract and 0.5 g/dl of NaCl, andcultured at 31.5° C. for 20 hours. One platinum loop of the obtainedcells were inoculated into a medium containing 3 g/dl of glucose, 6.5g/dl of ammonium sulfate, 0.1 g/dl of KH₂PO₄, 0.04 g/dl of MgSO₄, 0.001g/dl of FeSO₄, 0.001 g/dl of MnSO₄, 300 μg/dl of vitamin B₁, 200 μg/dlof biotin and soybean protein hydrolysate (165 mg/dl as N amount) andadjusted to pH 7.0 with NaOH,.and cultured at 31.5° C. for 24 hours asseed culture.

The above seed culture broth was. inoculated in an amount of 1 ml into amedium containing 4 g/dl of glucose, 6.5 g/dl of ammonium sulfate, 0.5g/dl of KH₂PO₄₁ 0.04 g/dl of MgSO₄, 0.001 g/dl of FeSO₄, 0.01 g/dl ofMnSO₄, 5 μg/dl of vitamin B₁, 5 μg/dl of biotin and soybean proteinhydrolysate (45 mg/dl as N amount) and adjusted to pH 7.0 with KOH, andcultured in a flask at 31.5° C. for 50 hours with shaking. Accumulationamount of L-arginine (concentration, mg/dl) in culture broth of eachstrain was measured. The results are shown in Table 2. As a result, theargR-disrupted strain accumulated L-arginine in a markedly larger amountcompared with the parent strain. TABLE 2 L-Arginine accumulation amountStrain (mg/dl) AJ13029 20 AJ13029ΔR 120(Explanation of Sequence Listing)SEQ ID NO: 1: primer for amplification of kanamycin resistance gene ofStreptococcus faecalisSEQ ID NO: 2: primer for amplification of kanamycin resistance gene ofStreptococcus faecalisSEQ ID NO: 3: primer for amplification of vector portion of pHSG399SEQ ID NO: 4: primer for amplification of vector portion of pHSG399SEQ ID NO: 5: nucleotide sequence of replication control region of pSFK6SEQ ID NO: 6: amino acid sequence that may be encoded by ORF in pSFK6SEQ ID NO: 7: nucleotide sequence of replication control region of p48KSEQ ID NO: 8: amino acid sequence that may be encoded by ORF in p48KSEQ ID NO: 9: primer for 1st PCR for introducing mutation of 1534th C toT into pSFK6SEQ ID NO: 10: primer for 1st PCR for introducing mutation of 1534th Cto T into pSFK6SEQ ID NO: 11: primer for 1st PCR for introducing mutation of 1534th Cto T into pSFK6SEQ ID NO: 12: primer for 1st PCR for introducing mutation of 1534th Cto T into pSFK6SEQ ID NO: 13: primer for 2nd PCR for introducing mutation of 1534th Cto T into pSFK6SEQ ID NO: 14: primer for 2nd PCR for introducing mutation of 1534th Cto T into pSFK6SEQ ID NO: 15: primer for argR gene amplification SEQ ID NO: 16: primerfor argR gene amplificationSEQ ID NO: 17: nucleotide sequence of DNA fragment containing argR geneSEQ ID NO: 18: amino acid sequence that may be encoded by the above DNAfragmentSEQ ID NO: 19: primer for argR gene amplificationSEQ ID NO: 20: primer for argR gene amplificationSEQ ID NO: 21: primer for amplifying portions other than argR gene ORFof plasmid containing argR geneSEQ ID NO: 22: primer for amplifying portions other than argR gene ORFof plasmid containing argR gene

1.-3. (canceled)
 4. A method for producing L-arginine, which comprisesculturing a coryneform bacterium in which an ar inine repressor does notfunction in a normal manner and which has L-arginine producing abilityin a medium to produce and accumulate L-arginine in the medium, andcollecting the L-arginine from the medium.
 5. The method of claim 4,wherein a polynucleotide encoding the argR arginine repressor on achromosome of said coryneform bacterium is disrupted, and saidpolynucleotide encoding the argR arginine repressor prior to beingdisrupted has the nucleotide sequence shown in SEQ ID NO: 17, and saidisolated coryneform bacterium over produces L-arginine as compared to anunmodified coryneform bacterium.
 6. The method of claim 5, wherein saidcoryneform bacterium belongs to a species selected from the groupconsisting of Corynebacterium acetoacidophilum, Corynebacteriumacetoglutamicum, Corynebacterium alkanolyticum, Corynebacteriumcallunae, Corynebacterium glutamicum, Corynebacterium lilium,Corynebacterium melassecola, Corynebacterium thermoaminogenes,Corynebacterium herculis, Brevibacterium divaricatum, Brevibacteriumflavum, Brevibacterium immariophilum, Brevibacterium lactofermentum,Brevibacterium roseum, Brevibacterium saccharolyticum Brevibacteriumthiogenitalis, Brevibacterium album, Brevibacterium cerinum, andMicrobacterium ammoniaphilum.
 7. The method of claim 5, wherein saidcoryneform bacterium is resistant to a compound selected from the groupconsisting of sulfa drugs, 2-thiazolealanine, andαa-amino-β-hydroxyvaleric acid.
 8. The method of claim 5, wherein saidcoryneform bacterium exhibits auxotrophy for a compound selected fromthe group consisting of L-histidine, L-proline, L-threonine,L-isoleucine, L-methionine, and L-tryptophan.
 9. The method of claim 5,wherein said coryneform bacterium is resistant to a compound selectedfrom the group consisting of ketomalonic acid, fluoromalonic acid, andmonofluoroacetic acid.
 10. The method of claim 5, wherein saidcoryneform bacterium is resistant to a compound selected from the groupconsisting of arginol and X-guanidine, wherein X is derived from a fattyacid or aliphatic chain.
 11. The method of claim 5, wherein saidcoryneform bacterium belongs to a genus selected from the groupconsisting of the genus Corynebacterium, the genus Brevibacterium, andthe genus Microbacterium.
 12. The method of claim 5, wherein saidcoryneform bacterium is a recombinant coryneform bacterium.
 13. Themethod of claim 5, wherein said polynucleotide encoding the argRarginine repressor prior to being disrupted encodes the amino acidsequence shown in SEQ ID NO: 18 or said polynucleotide encoding the argRarginine repressor prior to being disrupted is obtained from chromosomalDNA of the bacterium by PCR with oligonucleotide primers having anucleotide sequence shown in SEQ ID NO: 15 and SEQ ID NO:
 16. 14. Themethod of claim 13, wherein said coryneform bacterium belongs to aspecies selected from the group consisting of Corynebacteriumacetoacidophilum, Corynebacterium acetoglutamicum, Corynebacteriumalkanolyticum, Corynebacterium callunae, Corynebacterium glutamicum,Corynebacterium lilium, Corynebacterium melassecola, Corynebacteriumthermoaminogenes, Corynebacterium herculis, Brevibacterium divaricatum,Brevibacterium flavum, Brevibacterium immariophilum, Brevibacteriumlactofermentum, Brevibacterium roseum, Brevibacterium saccharolyticumBrevibacterium thiogenitalis, Brevibacterium album, Brevibacteriumcerinum, and Microbacterium ammoniaphilum.
 15. The method of claim 13,wherein said coryneform bacterium is resistant to a compound selectedfrom the group consisting of sulfa drugs, 2-thiazolealanine, andα-amino-β-hydroxyvaleric acid.
 16. The method of claim 13, wherein saidcoryneform bacterium exhibits auxotrophy for a compound selected fromthe group consisting of L-histidine, L-proline, L-threonine,L-isoleucine, L-methionine, and L-tryptophan.
 17. The method of claim13, wherein said coryneform bacterium is resistant to a compoundselected from the group consisting of ketomalonic acid, fluoromalonicacid, and monofluoroacetic acid.
 18. The method of claim 13, whereinsaid coryneform bacterium is resistant to a compound selected from thegroup consisting of arginol and X-guanidine, wherein X is derived from afatty acid or aliphatic chain.
 19. The method of claim 13, wherein saidcoryneform bacterium belongs to a genus selected from the groupconsisting of the genus Corynebacterium, the genus Brevibacterium, andthe genus Microbacterium.
 20. The method of claim 13, wherein saidcoryneform bacterium is a recombinant coryneform bacterium.